The HIV-1 envelope (env) glycoprotein gp12O and its biosynthetic precursor gpl6O play a critical role in the pathophysiology of HIV-1 infection and are principal target antigens for HIV-1 vaccines. The long term goal of this project is to understand the recognition of these proteins by human T cells. During the initial funding period, env-specific human CD8 + and CD4 + cytolytic T lymphocytes (CTL) clones were used to analyze the mechanisms by which the env proteins are processed for recognition by T cells. For both class I- and class II-restricted recognition, novel antigen processing pathways were identified. This proposal describes further studies of env protein processing designed to determine the precise structure of the naturally processed env peptides present on infected cells and to characterize the processing mechanisms that generate these peptides. With respect to env processing for recognition by CD8 + CTL, the proposed studies will extend recent work from this laboratory showing that the env protein is processed by a novel pathway which is localized to the endoplasmic reticulum (ER) and which does not depend on the transport proteins Tap-1 and Tap-2 previously thought to be essential for presentation of viral proteins. Naturally processed env peptides will be isolated from class I molecules of infected cells and sequenced to determine whether these peptides are generated by Tap-1/Tap-2-dependent (cytoplasmic) or Tap-1/Tap-2-independent (ER) processing reactions. The relationship between ER processing of the HIV-1 env protein and other ER proteolysis reactions will be evaluated and the hypothesis that targeting of the env protein for ER degradation can enhance processing and presentation of env epitopes will be tested. In addition, studies are proposed to determine whether this novel pathway is utilized by all viral envelope proteins. With respect to env processing for recognition by CD4 + T cells, the proposed studies will extend work form this laboratory showing that endogenously synthesized env protein is directly processed in infected cells for association with class II MHC molecules. The hypothesis that this processing reaction operates on env protein internalized from the cell surface will be tested by isolating and sequencing naturally processed env peptides from the class II molecules of infected cells and by comparing their structures with those generated by processing of exogenous env protein. Studies with env mutants will address whether env processing is inhibited by mutations that alter potential internalization signal sequences or enhanced by mutations that target env protein to lysosomes. In addition to providing basic information about antigen processing, these studies may facilitate the design of new vaccines that elicit an "optimized" T cell response specific for the exact antigenic peptides expressed on infected cells.